CDK9 inihibitors for use in the treatment of cancer

Abstract

There is disclosed an inhibitor of a protein for use in the treatment of cancer.

Description

[0001] "CDK9 INIHIBITORS FOR USE IN THE TREATMENT OF CANCER"

[0002] Cross-Reference To Related Applications

[0003] This patent appl ication claims priority from Italian patent application no . 102024000003478 filed on February 19 , 2024 , the entire disclosure of which is incorporated herein by reference .

[0004] Technical Field of the Invention

[0005] The present invention relates to an inhibitor of a protein comprising SEQ ID NO : 1 , speci fically referred to as CDK9-55 in the following, for use in treating cancer .

[0006] Prior Art

[0007] DNA double-strand breaks ( DSBs ) contribute to genome instability, a key feature of cancer since it generates the genetic diversity that enables the acquisition of hallmark capabilities during tumour development and progression .

[0008] DNA double-strand breaks (DSBs ) are among the most toxic DNA lesions threatening genome stability . To tackle this threat , DSBs are repaired by two principal processes : homologous recombination (HR) and non-homologous end- j oining (NHEJ) . HR uses the sister chromatid as a template for faithful repair and requires DNA end-resection to generate long 3 ' single-stranded DNA ( ssDNA) to invade this template ; conversely, NHEJ j oins DNA ends irrespective of their original sequence and, thus , is error-prone .

[0009] Chemotherapeutic drugs often lead to damage of DNA in cells with subsequent induced resi stance of tumour cells to the chemotherapeutic . Many drugs , currently used in cancer therapy, work on the stability of the DNA double helix leading to DNA damage , such as topoisomerase inhibitors , alkylating agents , and so on . One of the problems with these cancer treatments are the evolving resistance mechanisms that reduced the ef ficacy of the tumour therapy . To this aim, the identi fication of new molecular targets for cancer treatments can be useful to bypass the emerging resistance mechanisms .

[0010] The need is felt in the art for ef ficient means to obtain error-prone DNA repair (NHEJ) instead of an error- free DNA repair (HR) .

[0011] Summary of the Invention

[0012] It is an obj ect of the present invention to provide a molecule that can guide DNA repair in the direction of NHEJ instead of HR, in particular for use in assessing responsiveness of a tumour to a chemotherapeutic drug and for use in the treatment of cancer. In particular, in the context of cancer therapy, the increase of NHEJ activity could be essential to expand the mutational rate of cancer, leading to a major sensitivity to the therapy.

[0013] This obj ect is achieved by means of the inhibitor for the use as defined in claim 1 , the composition for the use as defined in claim 7 and the use of the protein as defined in claim 16 .

[0014] The present inventors demonstrated that the CDK9 knockout (KO) induces a reduction of fidelity DNA repair (HR) favouring an error- prone NHEJ mechanism. In particular, human cervical carcinoma cells (HeLa cells) KO for CDK9 protein showed an increased sensibility to the currently used cancer therapy drugs with respect to HeLa control, indicating that the identification of new protein kinase inhibitors is useful as a combination strategy for cancer treatment . Brief Description of the Drawings

[0015] Figure 1A shows Western blot analysis of HeLa cells transfected with siRNA CDK9 (siCDK9) , or control (siCTR) , and incubated for 24 hours followed by transfection with the single CDK9 isoforms (pCEFLHA CDK9 55 kDa and pCEFL HA CDK9 42kDa) and by transfection with pCEFL HA control plasmid, incubated for additional 48 hours. At the end of incubation time, HeLa cells were treated with 1 pM camptothecin (CRT) , or not treated, for 2 hours. RPA32 was used as a DNA damage control and Lamin A / C as a protein loading control.

[0016] Figure IB shows immunofluorescence analysis of HeLa cells transfected with siRNA CDK9 (siCDK9) , or control (siCTR) , and incubated for 24 hours followed by transfection with the single CDK9 isoforms (pCEFLHA CDK9 55 kDa and pCEFL HA CDK9 42kDa) and by transfection with pCEFL HA control plasmid, followed by CPT treatment with 1 pM CPT for 2 hours. RPA32 foci intensity was measured by Fiji software. More than 30 cells were analysed for each condition in 3 independent experiments; data represent means ± standard deviation. Data were subjected to one-way repeated measures ANOVA with Kruskal-Wallis post-test, to compare all groups. Statistically significant differences are indicated with: **very significant (P < 0.01) and ***extremely significant (P < 0.001)

[0017] Figure 1C shows immunofluorescence analysis of HeLa cells transfected with siRNA CDK9 (siCDK9) , or control (siCTR) , and incubated for 24 hours followed by transfection with the single CDK9 isoforms (pCEFLHA CDK9 55 kDa and pCEFL HA CDK9 42kDa) and by transfection with pCEFL HA control plasmid, followed by CPT treatment with 1 pM CPT for 2 hours. BRCA1 foci intensity was measured by Fiji software. More than 30 cells were analyzed for each condition in 3 independent experiments; data represent means ± standard deviation. Data were subjected to one-way repeated measures ANOVA with Kruskal-Wallis post-test, to compare all groups. Statistically significant differences are indicated with: ***P-value <0.001.

[0018] Figure ID shows immunofluorescence analysis of HeLa cells transfected with siRNA CDK9 (siCDK9) , or control (siCTR) , and incubated for 24 hours followed by transfection with the single CDK9 isoforms (pCEFLHA CDK9 55 kDa and pCEFL HA CDK9 42kDa) and by transfection with pCEFL HA control plasmid, followed by CPT treatment with 1 pM CPT for 2 hours. RIF1 foci intensity was measured by Fiji software. More than 30 cells were analysed for each condition in 3 independent experiments; data represent means ± standard deviation. Data were subjected to one-way repeated measures ANOVA with Kruskal-Wallis post-test, to compare all groups. Statistically significant differences are indicated with: **P-value < 0.01 and ***P-value <0.001.

[0019] Figure 2A shows Western blots with an antibody against 950 CDK9 in wild-type (wt) HeLa cells and CDK9-55KO clones to check the CDK9-55 knockout efficiency. GAPDH was analyzed as a loading control.

[0020] Figure 2B shows Western blots of Wt HeLa cells and CDK9- 55KO clones (cl2 and cl4) which were either treated with IpM CPT for 2 hours or not treated (NT) . Western blot was carried out with antibodies against CDK9, phosphorylated RPA 32 (pRPA32 S4 / 8) , total RPA 32, and lamin A / C, latter both used as a loading control. The black arrow indicates the phosphorylated RPA32 bands.

[0021] Figure 2C shows results of the SMART (single-molecule analysis of resection tracks) assay, in which wt HeLa and cl2 cells were pulse-labeled with BrdU for 24 hours and then treated with IpM CPT, as above. We analyzed more than 200 fibers for each cell type in 3 independent experiments; data represent means ± standard deviation. Data were subjected to multiple comparisons with Mann-Whitney to compare 2 groups. Statistically significant differences are indicated with ***P-value <0.001.

[0022] Figure 2D shows Western blots of HeLa and CDK9 55KO cells which were treated, or not (NT) , with IpM CPT for 2 hours followed by chromatin enriched purification. The soluble (S) and chromatin enriched cell fractions (P) were analyzed by western blotting through the indicated antibodies. Total RPA32 was used as DNA damage control and lamin A / C as a loading control.

[0023] Figure 2E shows graphs which represent the percentage of colonies formation through clonogenic assay. Wt HeLa cells and CDK9-55KO clones were treated with Olaparib at crescent concentrations (1.5-2-2.5 pM) and, ten days after treatment, stained with crystal violet. Data represent means ± standard deviation (n = 3 independent experiments) . Data were subjected to one-way repeated measures ANOVA with Dunnett's post-test to compare all groups versus control (wt) . Statistically significant differences are indicated with: *P-value <0.05, **P-value <0.01. Figure 2F shows graphs which represent the percentage of colonies formation through clonogenic assay. Wt HeLa cells and CDK9-55KO clones were treated with CTP at crescent concentrations (2.5-5-7 nM) and, ten days after treatment, stained with crystal violet. Data represent means ± standard deviation (n = 3 independent experiments) . Data were subjected to one-way repeated measures ANOVA with Dunnett's post-test to compare all groups versus control (wt) . Statistically significant differences are indicated with: *P-value <0.05, **P-value <0.01.

[0024] Figure 3A shows SMART (single-molecule analysis of resection tracks) assay in wt HeLa cells and CDC23 (S588A) - mutated cl9 pulse-labeled with BrdU for 24 hours and then treated with IpM camptothecin (CPT) for 2 hours. More than 200 fibers for each cell type in 3 independent experiments; data represent means ± standard deviation. Data were subjected to an unpaired Student t-test. Statistically significant differences are indicated with ***P-value <0.001.

[0025] Figure 3B shows the results of FACS analysis of BrdU incorporation in non-denaturing conditions to evaluate single-stranded DNA formation in wt HeLa and cl9, treated or not treated (NT) with IpM camptothecin (CPT) for 2 hours. More than 200 fibers for each cell type in 3 independent experiments; data represent means ± standard deviation. Data were subjected to an unpaired Student t-test. Statistically significant differences are indicated with ***P-value <0.001.

[0026] Figure 3C shows results of the SMART (single-molecule analysis of resection tracks) assay of HeLa cells and cl9 which were treated with IpM CPT as described above and analysed by immunofluorescence with an anti-RAD51 antibody. RAD51 foci intensity was measured by Fiji software. Data represent means ± standard deviation (n = 3 independent experiments) . Data were subjected to multiple comparisons with Mann-Whitney to compare 2 groups. Statistically significant differences are indicated with: ***P-value <0.001.

[0027] Figure 3D shows the results obtained when HeLa wt cells and CDK 9 55KO clones (cl2 and cl4) were treated with CPT at indicated drug concentration for 72 hours followed by SRB cell viability assay. Data represent means ± standard deviation (n = 3 independent experiments) . Data were subjected to one-way repeated measures ANOVA with Dunnett's post-test to compare all groups versus control (wt) . Statistically significant differences are indicated with: *P-value <0.05, **P-value <0.01, ***P-value <0.001.

[0028] Figure 3E shows a cell survival assay upon Olaparib treatment in HeLa wt and CDK 9 55KO cell clones (cl2 and cl4) was carried out as in D. Data represent means ± standard deviation (n = 3 independent experiments) . Data were subjected to one way repeated measures ANOVA with Dunnett's post-test to compare all groups versus control (wt) . Statistically significant differences are indicated with: **P-value <0.01, ***p value <0.001.

[0029] Figure 4A shows proliferation assays with f lavopiridol . Figure 4B shows proliferation assays with AZD4573.

[0030] Detailed Description of the Invention Cycl in- dependent kinases ( CDKs ) , which are serine / threonine kinases whose function depends on the interaction with cyclin regulatory subunits , are involved in cell processes , such as transcription, DNA damage response ( DDR) , cell death, di f ferentiation, immune response , and metabolism . CDK9 exists in two isoforms , CDK9-42 ( 42 kDa ) and CDK9-55 ( 55 kDa ) , which originate from di f ferent promoters separated by around 500 bp, and with CDK9-55 having 117 additional amino acids in its N-terminal portion .

[0031] The present inventors have found that an isoform of the multi functional cyclin-dependent kinase 9 , CDK9-55 , plays a role in af fecting the repair pathway choice by favoring HR .

[0032] The present inventors have also identi fied the mechanism by which CDK9-55 can act is the phosphorylation of CDC23 in position Ser588 . This amino acid is one of the most frequently modi fied amino acids in response to chemotherapeutic drugs in dif ferent tumors . It was previously unknown which protein kinase was responsible for phosphorylation of Ser588 of CDC23 .

[0033] A pharmacological inhibitor of the phosphorylation of Ser588 of CDC23 can therefore increase the sensitivity of cells to DNA damage induced for example by camptothecin, thus allowing the reduction of doses of the chemotherapeutic drug and its side ef fects .

[0034] The present invention therefore relates to an inhibitor of a protein comprising SEQ ID NO: 1 for use in the treatment of cancer .

[0035] The inhibitor preferably impairs CDC23 Ser588 phosphorylation.

[0036] The inhibitor is preferably a small molecule, more preferably a small molecule selected from the group consisting of AZD 4573 and flavopiridol, even more preferably AZD 4573.

[0037] Alternatively, the inhibitor of CDK9-55 is preferably an siRNA directed to CDK9-55.

[0038] Preferably, the inhibitor enhances effectiveness of the chemotherapeutic drug on a tumour. In particular, it enhances sensitivity of the tumour to the chemotherapeutic drug. The chemotherapeutic drug is preferably a DNA-damaging chemotherapeutic or a PARP inhibitor.

[0039] The protein coirprising SEQ ID NO: 1 is encoded by the nucleic acid, which comprises SEQ ID NO: 2.

[0040] The present invention also relates to a composition comprising an inhibitor of a protein comprising SEQ ID NO: 1 and a chemotherapeutic drug for use in the treatment of cancer. The inhibitor preferably impairs CDC23 Ser588 phosphorylation.

[0041] Preferred inhibitors of the protein coirprising SEQ ID NO: 1 (CDK9- 55) are small molecules, preferably selected from the group consisting of AZD 4573 and flavopiridol, or siRNA directed to CDK9-55.

[0042] The present invention also relates to the use of a protein comprising SEQ ID NO: 1 in assessing responsiveness of a tumour to a chemotherapeutic drug.

[0043] Examples

[0044] The role of an isoform of the multi functional cyclin- dependent kinase 9 , CDK9-55 ( SEQ ID NO : 1 ) , in DNA repair was investigated by generating CDK9-55-knockout HeLa clones ( through CRISPR-Cas 9 ) , which showed potential HR dys function . A phosphoproteomic screening in these clones treated with camptothecin revealed that CDC23 ( cell division cycle 23 ) , a component of the E3-ubiquitin ligase APC / C (anaphase promoting complex / cyclosome ) , is a new substrate of CDK9-55, with S588 being its putative phosphorylation site. Mutated non-phosphorylatable CDC23 (S588A) affected the repair pathway choice by impairing HR and favoring error- prone NHEJ. Moreover, CDC23 (S588A) promoted the ubiquitination of UFL1, a recently identified HR player.

[0045] These results show that CDK9-55 guides APC / C in choosing the correct DNA repair pathway, possibly by regulating UFL1 stability. CDK9 can therefore be used when designing CDK51 inhibitor-based cancer therapies.

[0046] Example 1

[0047] CDK9 protein was recently described to be involved in DNA damage response (DDR) and the discrimination between its two protein isoforms in DDR had never been performed. CDK9 proteins were silenced through siRNAs (siCDK9) followed by differential expression of CDK9 42 or 55 (Figure 1A) to characterize the response to camptothecin (CPT) . First of all, silencing of CDK9 reduces phosphorylation of Replication Protein A 32Kda (pRPA32) foci intensity and BRCA1, confirming its role in homologous recombination (HR) (Figure 1B-C) ; the overexpression of 42 or 55 isoforms recovers RPA32 phosphorylation in a similar manner but not for BRCA1 (Figure 1B-C) . To check if CDK9 silencing could affect the HR repair favouring the NHEJ, a Replication timing regulatory factor 1 (RIF1) immunofluorescence was performed (Zimmermann & de Lange, 2014) . As reported in figure ID, siCDK9 increased RIF1 foci in response to CPT in HeLa cells, followed by a reduction of its intensity with the differential overexpression of CDK9 isoforms (Figure ID) . Overall these data show the involvement of both CDK9 protein isoforms in DDR.

[0048] Example 2

[0049] To best describe the possible role of CDK9 isoforms in the HR, clones knockout for CDK9-55 (55KO cl2, cl3, and cl4) were generated without affecting CDK9-42 levels (Figure 2A) , through the CRISPR-CAS9 system, wt HeLa cells and 55KO clones were then treated with CRT analysing the expression of pRPA32 S4 / 8, and its impaired phosphorylation in 55KO clones compared with control cells was observed after 2 hours of treatment (Figure 2B) . Consistently, Single-Molecule Analysis of Resection Tracks assay (SMART) showed a marked reduction of resected DNA length in 55KO cl2 compared with wt HeLa cells upon CPT treatment (Figures 2C) . Finally, a chromatin loading of RAD51 recombinase (RAD51) was analysed showing an impaired loading in response to CPT (Figure 2D) . Moreover, clonogenic assays were performed with different doses of either Olaparib, or CPT, showing an increased sensitivity to both drugs in 55KO clones compared with control cells (Figures 2E, 2F) . These data suggest that CDK9- 55 knockout affects the DNA end-resection process, essential for HR.

[0050] Example 3

[0051] A global phosphoproteomic screening was performed in CPT-treated and untreated wt HeLa and 55KO cl2 to identify possible new CDK9-55 substrates involved in DDR, identifying CDC23 as a new target of CDK9-55 in DNA repair. HeLa cells expressing a mutated non-phosphorylatable CDC23, in which S588 was substituted with alanine (S588A) were generated by using the CRISPR-Cas 9-D10A system . A SMART as say was performed, after treatment with CRT , showing a reduced resected DNA length in CDC23 ( S588A) -mutated cl 9 cells compared with the control ( Figure 3A) . Moreover, FACS analysis of Bromodeoxyuridine (BrdU) incorporation in nondenaturing conditions , confirmed an impaired formation of resected ssDNA ( Figure 3B ) . One of the important steps of the HR is the strand invasion which is regulated by RAD51 ; the analysis of RAD51 foci , by immunofluorescence , showed a reduced signal in cl 9 ( Figure 3C ) . To check i f HeLa cells , carrying CDC23 S588A point mutation, could be synthetic lethal with Olaparib treatment , cell survival and clonogenic assays were performed, showing an increased sensitivity to Olaparib in CDC23 S588A cell clones compared with control cells ( Figures 3D and 3E ) .

[0052] Example 4

[0053] A new CDK9-55 consensus sequence was found, which led to the identi fication of the APC / C subunit CDC23 as a new CDK9-55 substrate guiding the DNA repair pathway choice , possibly by af fecting UFL1 ubiquitination . Considering the recently established role of CDKs in DDR, their inhibition has been proposed to sensiti ze cancers to DNA-damaging chemotherapeutics or PARP inhibitors . CDK9 could be a valuable target , especially in tumours with dysregulated transcriptional programs . These data add a new understanding to CDK9 functions , which should be considered when designing CDK-inhibition-based therapies .

[0054] Overall , the APC / C subunit CDC23 was identi fied as a new CDK9-55 substrate guiding the DNA repair pathway . Considering the recently established role of CDKs in DDR, their inhibition can sensitize cancers to DNA-damaging chemotherapeutics or PARP inhibitors. CDK9 is a valuable target, especially in tumors with dysregulated transcriptional program.

[0055] Example 5

[0056] Cell viability assays were performed on HeLa wild-type (wt) and CDK9 55KO cell lines treated with Flavopiridol or AZD4573. Flavopiridol is a CDK inhibitor currently being tested in clinical trials, and we assessed whether its use could reduce cell proliferation. As shown in the figure, Flavopiridol induced dose-dependent inhibition of cell proliferation in both HeLa wt and CDK9 55KO cell lines.

[0057] Additionally, the effect of AZD4573, a specific kinase inhibitor for CDK9 enzymatic activity, on cell proliferation was investigated in HeLa wt and 55KO cell lines. As shown in the figure, AZD4573 reduced proliferation in all cell lines. However, both kinase inhibitors demonstrated a reduced effect on the 55KO cell clones compared to HeLa wt cells, suggesting the potential use of CDK9 55KO as a target for cancer therapy.

Claims

CIAIMS1. An inhibitor of a protein coirprising SEQ ID NO: 1, for use in the treatment of cancer.

2. The inhibitor for use according to claim 1, wherein the inhibitor impairs CDC23 Ser588 phosphorylation.

3. The inhibitor for use according to claim 1 or 2, wherein the inhibitor is a small molecule.

4. The inhibitor for use according to claim 3, wherein the small molecule is selected from the group consisting of AZD 4573 and f lavopiridol .

5. The inhibitor for use according to claim 4, wherein the inhibitor is AZD 4573.

6. The inhibitor according to claim 1 or 2, wherein the inhibitor of CDK9-55 is an siRNA directed to CDK9-55.7 . A composition coirprising an inhibitor of a protein comprising SEQ ID NO: 1 and a chemotherapeutic drug for use in the treatment of cancer .

8. The composition for use according to claim 7, wherein the inhibitor impairs CDC23 Ser588 phosphorylation.

9. The composition for use according to claim 7 or 8, wherein the inhibitor is a small molecule .

10. The composition for use according to claim 9, wherein the small molecule is selected from the group consisting of AZD 4573 and f lavopiridol .

11. The composition for use according to claim 10, wherein the inhibitor is AZD 4573.

12. The composition for use according to claim 7 or 8, wherein the inhibitor of CDK9-55 is an siRNA directed to CDK9-55.

13. The composition for use according to any of claims 7 to 12,wherein the inhibitor enhances effectiveness of the chemotherapeutic drug on a tumour.

14. A composition for use according to any of claims 7 to 13, wherein the inhibitor enhances sensitivity of the tumour to the chemotherapeutic drug.

15. A composition for use according to any of claims 7 to 14, wherein the chemotherapeutic drug is a DNA-damaging chemotherapeutic or a PARP inhibitor.

16. Use of a protein comprising SEQ ID NO: 1 in assessing responsiveness of a tumour to a chemotherapeutic drug.

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